ABSTRACT Cassava is currently the second most important source of carbohydrates on the African continent. In the last two decades, cassava crops have been severely affected by outbreaks of cassava mosaic disease (CMD). South African cassava mosaic virus (SACMV) has been associated with CMD outbreaks in the Mpumalanga province. Advances in post-transcriptional gene silencing (PTGS) technology have provided promising new strategies for the engineering of virus resistance in plants. Inverted repeat (IR) constructs are currently the most potent inducers of PTGS, however, these constructs are inherently unstable. The purpose of this study was to develop IR constructs with an improved stability for the efficient induction of PTGS in plants. Two mismatched inverted repeat constructs, one targeting the SACMV BC1 open reading frame, the other targeting the Maize streak virus (MSV) AC1 open reading frame, were successfully created. Sodium bisulfite was used to deaminate cytosine residues on the sense arm of the constructs. The resulting number of GT mismatches was seemingly sufficient to stabilize the linear conformation of the IR constructs, as they were efficiently propagated by E.coli DH5!, and subsequently behaved like linear DNA molecules. Furthermore, it was found that the number of mismatches on the BC1 construct (17.5%) was ideal, as the subsequent stability of the predicted RNA hairpin was not affected. Due to the higher number of mismatches on the AC1 construct (23.5%), it was found that the loop region of the RNA hairpin was marginally destabilized. Despite this, long stretches of stable dsRNA were still produced from the AC1 IR construct, and is likely to induce PTGS. Interestingly, it was observed that the mismatched IR constructs, although still replicated in E.coli, were marginally destabilized in Agrobacterium. Therefore, it was deduced that the stability of a mismatched IR construct may be influenced by the particular intracellular environment of an organism. Due to the recalcitrance of cassava to transformation, a model plant system, Nicotiana benthamiana, was used to screen constructs for toxicity, stability, and efficiency of PTGS induction. Agrobacteriummediated transformation and regeneration of N. benthamiana was optimized, and 86% transformation efficiency was achieved when using leaf disk explants. It was found that the addition of an ethylene scrubber, potassium permanganate, substantially increased the rate of regeneration by reducing the frequency of hyperhydritic plants. Transgene iv integration was confirmed by PCR amplification of the hptII gene in the T-DNA region. Transgene expression was confirmed by screening for GUS and GFP reporter genes. No toxic responses to the transgene have been observed thus far. Studies are currently underway to confirm the stability of the mismatched IR constructs in N. benthamiana. PAGE Northern blotting is being done, as the detection of siRNAs derived from the transgene will confirm that constructs are functional. In addition, infectivity assays are underway to determine the efficacy of BC1 knockdown by a stably integrated construct. Due to the enhanced stability of mismatched IR constructs, they may be an appealing alternative to currently available intron-spliced, or exact matched hairpin systems.

Plant microRNAs play important roles in plant growth and development. Here we investigated the roles of miRNAs in the plant abiotic stress, development and viral infection. MicroRNA membrane array analysis using five different abiotic stress treatments resulted in the identification of 8 novel stress inducible miRNA-families. Functional studies on novel stress inducible miR168 revealed its functional relation with abiotic stress. Over expression of miR168 in Arabidopsis showed upregulation of four stress related miRNAs (miR163, miR167, miR398 and miR408). Analysis of 9 independent transgenic lines showed induction of miR398, an oxidative stress responsive miRNA with a corresponding down regulation of its target genes. Heavy metal oxidative stress tolerance bioassays confirmed the susceptibility of transgenics compared to the wild types indicating the fact that the miR168 is indirectly involved in plant abiotic stress by inducing other stress responsive miRNAs. MicroRNAs are highly conserved across the plant kingdom. A miRNA atlas was drafted for different tomato organs and fruit stages using the known miRNA sequences from different plants species. A large variation in both number and level of miRNA expression pattern was observed among different organs as well as among fruit stages. In the present investigation, we have found a window of expression for different miRNAs during the fruit development. A gradual decrease in the expression levels of miR160h, miR167a and miR399d and a gradual increase in miR164a have been noticed towards the fruit maturation while miR398b showed dual peaks during fruit development indicating a potential role of various miRNAs in fruit development and maturation. Sonchus yellow net virus (SYNV) infected Nicotinana benthamiana leaves showed severe disease symptoms at two weeks post infection (WPI) and gradually recovered from the SYNV infection after 4-5 WPI correlating with the overall miRNA levels. The miRNA array and northern analysis showed an overall reduction of miRNA biogenesis during 2WPI followed by restoration to normal levels supporting the idea that the SYNV indeed interfered with the host miRNA levels which caused the symptoms and recovery phenotypes. Overall studies on plant abiotic stress, development and viral infection showed important roles of miRNAs in different aspects of plant life.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13017号 / 論農博第2827号 / 新制||農||1042(附属図書館) / 学位論文||H28||N4964(農学部図書室) / 32945 / 京都大学大学院農学研究科農学専攻 / (主査)教授 土井 元章, 教授 裏出 令子, 教授 奥本 裕 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

ARGONAUTE proteins (AGOs) are generally accepted as key components of the post transcriptional gene silencing mechanism, also involved in plant antiviral defense. Except for reports on the antiviral roles of AGO1, AGO2 and AGO7 in Arabidopsis, the exact roles played by the individual AGOs in other plant species are largely unknown. This research focused on the identification and characterization of AGOs involved in antiviral RNAi response to various viruses in N. benthamiana. Based on the temporal and spatial distribution of AGO transcripts in 3 and 8-week old plant root, stem and leaf tissues, expressions of NbAGO mRNAs were found to vary with age and tissue specificity. Plant endogenous AGO mRNAs were knocked down through virus induced gene silencing techniques using the Tobacco rattle virus vector system and posteriorly challenged with a GFP-chimeric virus construct deficient of a silencing suppressor. Unlike in control non-silenced plants, the Tomato bushy stunt virus construct deficient of its P19 silencing suppressor was consistently seen to exhibit a strong fluorescence on N. benthamiana plants silenced for NbAGOs 2 and X. Similar results were also obtained upon silencing of NbAGO2 using hairpin vector techniques. Comparable observations were also made when Tobacco mosaic virus GFP constructs were agroinfiltrated on NbAGO2 silenced plants further hinting the antiviral defense roles played by these AGOs. Agroinfiltration of Foxtailmosaic virus, Sunnhemp mosaic virus, and Turnip crinkle virus GFP chimeric constructs on NbAGO2 silenced N. benthamiana plants, however did not result in accumulation of GFP indicating the AGO antiviral defense specificity to TBSV and TMV. The results also hinted at a role for AGO7. Collectively my findings suggest that the expression of AGOs in N. benthamiana is tissue and age dependent, and that unlike in the model plant Arabidopsis where the main antiviral AGO is thought to be AtAGO1; in N. benthamiana, NbAGOs 2 and X seem to be involved in an antiviral defense role against TBSV and TMV with other AGOs perhaps contributing.

The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.

Le virus des nervures jaunes et nécrotiques de la betterave (Beet necrotic yellow vein virus, BNYVV) est l’agent infectieux responsable de la rhizomanie de la betterave sucrière, une maladie caractérisée par une prolifération anarchique du chevelu racinaire. Le Beet soil-borne mosaic virus (BSBMV) appartient également au genre Benyvirus mais n’est retrouvé qu’en Amérique du Nord. Ce virus, identifié pour la première fois au Texas, est morphologiquement et génétiquement semblable au BNYVV mais sérologiquement éloigné. Compte tenu des différences moléculaires existant, le BSBMV et BNYVV correspondent à deux espèces virales distinctes. Mon projet de thèse a consisté à étudier les interactions moléculaires entre le BNYVV et le BSBMV et rechercher les mécanismes impliqués dans la pathogénicité de ces deux virus. Des clones complets cDNA infectieux du BNYVV étaient disponibles, tout comme ceux de BSBMV. Compte tenu de l’aspect versatile de l’obtention de transcrits infectieux de ces différents clones, j’ai entrepris de produire des clones cDNA de chacun des ARN viraux sous contrôle d’un promoteur constitutive végétal pour initier l’infection par agroinfiltration. Les plantes hôtes Chenopodium quinoa et Nicotiana benthamiana ont été inoculées par des transcrits et agroinfiltrées pour initier l’infection virale et étudier l’interaction entre les ARN génomiques 1 et 2 des deux virus et étudier les propriétés de constructions chimères. En parallèle à ce travail, j’ai réalisé la caractérisation du suppresseur de RNA silencing du BSBMV en le comparant à celui du BNYVV. / The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.

La tristeza es la enfermedad viral más grave del cultivo de los cítricos y su agente causal es el virus de la tristeza de los cí­tricos (CTV). CTV induce uno o más de los siguientes sí­ndromes: I) decaimiento y muerte de los naranjos dulces (ND), pomelos y mandarinos injertados sobre el patrón naranjo amargo (NA), sí­ndrome conocido como "tristeza", II) enanismo, acanaladuras en la madera y fruta de pequeño calibre (stem pitting, SP), y III) enanismo y amarillamiento de plantas de semilla de limonero, pomelo y NA (seedling yellows, SY). La gama de huéspedes de CTV es muy restringida y hasta hace poco no se conocí­a ningún huésped herbáceo experimental. Actualmente, se sabe que la agroinfiltración de Nicotiana benthamiana, con clones de DNA complemantario (cDNA) del aislado T36 de CTV produce la infección sistémica de la planta, acompañada de sí­ntomas similares a los inducidos en cí­tricos, si bien la infección no queda limitada al floema. El aislado T36 induce SY y SP de lima Mejicana (LM), pero no en otros huéspedes como pomelo o ND. El estudio de los determinantes genéticos responsables de la inducción del sí­ndrome de SP requerí­a desarrollar un sistema genético basado en clones agroinfecciosos de un aislado inductor de estos sí­ntomas, como el aislado español T318A. Para ello, se partió de clones de cDNA de longitud completa de T318A previamente desarrollados en el laboratorio, capaces de replicarse en hojas de N. benthamiana pero incapaces de inducir infección sistémica y que presentaban varias mutaciones en su proteína de cápsida minoritaria p27. La corrección de dichas mutaciones y la construcción de nuevos clones de longitud completa de T318A marcados con el gen gfp, mostraron una correcta replicación en hojas agroinfiltradas de N. benthamiana, pero resultaron incapaces de inducir infección sistémica en este huésped experimental. La respuesta diferencial de N. benthamiana frente a distintas cepas de CTV permite estudiar los factores implicados en la interacción virus-huésped. Se analizó la interacción de las proteí­nas virales p20 y p25 de los aislados T36 y T318A con proteí­nas de N. benthamiana utilizando un abordaje consistente en: i) la expresión transitoria de p20/p25 marcadas con una etiqueta Strep-Tag en hojas de N. benthamiana, ii) purificación de los complejos proteí­na CTV-proteína huésped y análisis interactómico de los datos, y iii) estudio de la interacción directa entre p20/p25 y proteínas seleccionadas del huésped mediante análisis del doble hibrido en levadura y complementación bimolecular de fluorescencia (BIFC). Este abordaje proteómico mostró claras diferencias entre aislados que pueden explicar, en parte, el comportamiento diferencial de los aislados T36 y T318A en dicho huésped experimental. La inducción el síndrome de decaimiento por parte de CTV ha obligado a utilizar patrones tolerantes al decaimiento. Dichos patrones son menos adecuados. Las plantas de cí­tricos propagadas sobre NA e infectadas por CTV muestran necrosis en los tubos cribosos y disminución del floema funcional. Éstos desórdenes podrí­an ser consecuencia de la activación de los mecanismos de defensa como la reacción de hipersensibilidad desencadenada por la ruta del ácido salicí­lico o el silenciamiento génico mediado por RNA (post-transcriptional gene silencing, PTGS). Con el objetivo de avanzar en el mecanismo molecular de la resistencia del NA a la infección por CTV, se estudió el papel de diferentes genes de la planta implicados en las rutas mediante el uso de un vector viral basado en el genoma del virus del manchado foliar de los cítricos (citrus leaf blotch virus, CLBV). El silenciamiento génico de las rutas del AS o del PTGS en plantas NA y la inoculación de tres aislados de CTV patogénicamente diferentes mostró la implicación de ambas rutas en la defensa del NA frente a CTV. / Tristeza is the most important viral disease affecting citrus plants and Citrus tristeza virus (CTV) is the causal agent of this disease. CTV induces at least one of this syndromes: I) decline and death of sweet orange (SwO), grapefruits and mandarin trees grafted on sour orange (SO) rootstock, this syndromes is known as "tristeza", II) stunting, stem pitting (SP) and small fruits, and III) stunting and leaf chlorosis of lemon, grapefruit and SO seedlings (seedling yellows, SY). The host range of CTV is restricted and until recently no experimental herbaceous host was known. The agroinoculation Nicotiana benthamiana with clones of complementary DNA (cDNA) from the CTV isolate T36 cause the systemic infection of the plant and similar symptoms to those observed in citrus, although the infection is not limited to the phloem. T36 isolate induces SY and SP of Mexican lime (ML), but not in other hosts such as grapefruit and SwO. Therefore, to study the genetic determinants responsible of the SP syndrome induction was necessary to develop a genetic system based on agroinoculated clones from an isolate able to induce these symptoms, such as the Spanish isolate T318A. To do this, full length cDNA clones from T318A were obtained. They are able to replicate in N. benthamiana leaves but unable to induce systemic infection and showed several mutations in their protein of the minor coat, p27. The correction of these mutations and the construction of new clones of complete length from T318A labeled with the gfp gene, showed a proper replication in agroinoculated leaves of N. benthamiana, but they were still unable to induce systemic infection in this experimental host. The differential response of N. benthamiana to different CTV strains allows the study of the potential factors involved in the virus-host interaction. The aim of this work was study the interaction between the viral proteins p20 and p25 from the isolates T36 and T318A with N. benthamiana proteins with an analysis consisted in: I) the transitory expression of p20/p25 fused to Strep-Tag in N. benthamiana leaves, II) purification of the CTV protein-host protein complex and interatomic analysis of the data, and III) the study of the direct interaction between p20/p25 and selected plant proteins by the analysis of the double hybrid in yeast and bimolecular complementation of fluorescence (BIFC). The proteomic analysis showed strong differences between isolates that may partially explain the differential behavior of the T36 and T318A isolates in this experimental host. The induction of decline syndrome by CTV in citrus has leaded the use of tolerant rootstocks to decline. However, the use of such rootstocks is less suitable. Citrus plants propagated on SO rootstock and infected by CTV show phloem necrosis below the bud union that reduces the flow of carbohydrates to the roots. These symptoms may be a consequence of the activation of defense pathways in the plant, such as the hypersensitive reaction, hormone salicylic acid (SA) pathways or the RNA mediated post-transcriptional gene silencing (PTGS). Their relation is essential to know their implication in the decline. Therefore, the role of different genes involved in SA and PTGS has been studied by the silencing of plant genes using a viral vector (VIGS) based in the genome of the citrus leaf blotch virus (CLBV). The gene silencing of the SA and PTGS in SO and the inoculation of three different pathogenicity CTV isolates showed that both pathways are involved in the SO defense against CTV. The analysis of the proteins p20, p23 and p25 as possible suppressors of the AS indicating that the more virulent CTV isolates possess the more powerful suppressors. / La Tristesa és la malaltia viral més greu del cultiu dels cítrics. CTV induïx un o més de les sí­ndromes següents: I) decaïment i mort de taronger dolç§ (ND), pomelo i mandariner empeltats sobre el patró taronger amarg (NA), sí­ndrome conegut com "Tristesa", II) nanisme, estries en la fusta i fruita de xicotet calibre (SP) i III) nanisme i tonalitat groguenta de plantes de llavor de llimera, pomelo i taronger amarg (SY). El rang d'hostes de CTV és molt restringit i fins fa poc no es coneixia cap hoste herbaci experimental. Actualment es sap que la infecciò sistèmica en Nicotiana benthamiana amb clons de DNA complementari (cDNA) de l`aïllat de T36 provoca la infecció sistemàtica de la planta, acompanyada de síntomes similars als induïts en cí­trics, si be la infecció no queda llimitada al floema. L' aïllat T36 induïx SY i estries en la fusta de Llima Mexicana (LM), però no en altres hostes com a pomelo, ND o NA, l'estudi dels determinants genètics responsables de la inducció de la síndrome de SP requeria desenvolupar un sistema genètic basat en clons agroinfecciosos d'un aïllat inductor d'estos sí­mptomes, com l'aïllat espanyol T318A. Per a això, es va partir de clons de cDNA longitud completa de T318A prèviament desenvolupats al laboratori, capaços de replicar-se en fulls de N. benthamiana però incapaços d'induir infecció sistèmica i que presentaven varies mutacions en la seua proteïna de càpsida minoritatia p27. La correcció d`aquestes mutacions i la construcció de nous clons T318A de longitud completa marcats amb el gen gfp, van mostrar una correcta replicació en fulls agroinfiltradas de N. benthamiana però van resultar incapaços d'induir infecció sistèmica en aquest hoste experimental. La resposta diferencial dependent d'aïllat en N. benthamiana front CTV permet estudiar els possibles factors de la interacció virus- hoste. Es va dur a terme l'estudi de la funció de les proteínes virals p20 i p25 dels aïllats T36 i T318A amb proteïnes de N. benthamiana utilitzant un abordatge consistent en: i) l' expressió transitòria de les dues proteïnes p20/p25 marcades amb una etiqueta Strep-Tag en fulls de N. benthamiana, ii) purificació dels complexos proteïna CTV-proteïna hoste i anàlisi interactómic de les dades, i iii) estudi de la interacció directa per mitjà  de doble híbrid en llevat i complementació bimolecular de fluorescència (BIFC) de les proteïnes virals i determinades proteïnes de N. benthamiana. Aquest abordatge proteòmic va mostrar clares diferències entre aïllats que poden explicar el comportament diferencial dels aïllats T36 i T318A en aquest hoste experimental. La inducció de la sí­ndrome de decaïment per part de CTV en cí­trics ha obligat la utilització de patrons tolerants al decaïment. No obstant, aquestos patrons són agronòmicament menys adequats. Les plantes de cítrics propagades sobre NA i infectades por CTV mostren necrosi als tubs cribosos i disminució del floema funcional. Aquestos sí­mptomes poden ser conseqüència de l'activació de les rutes de defensa de la planta com la reacció d'hipersensibilitat, desencadenada per la ruta de l'àcid salicí­lic o el silenciamient gènic mediat per RNA (PTGS). Amb l'objectiu d'analitzar la implicació d¿aquestes rutes en la defensa, es va estudiar el paper de diferents gens implicats en la ruta de l'AS i del PTGS per mitjà  del silenciamient gènic induït per virus basat en el genoma del tacat foliar dels cítrics (CLBV). El silenciamient gènic de les rutes AS o PTGS en plantes NA i la inoculació de tres aïllats de CTV patogènicament diferents va mostrar la implicació de les dues rutes en la defensa del NA front CTV. L'analisis de les proteïnes p20, p23 i p25 com a possibles supressors de la ruta de l'AS va indicar que els aïllats més virulents de CTV posseïxen supressors més potents. / Gómez Muñoz, N. (2017). ESTUDIO DE LA INTERACCIÓN DIFERENCIAL ENTRE AISLADOS DEL VIRUS DE LA TRISTEZA DE LOS CÍTRICOS (CTV) Y SUS HUÉSPEDES [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/94624

Chez les eucaryotes, plusieurs protéines liant l'ARN ou RBPs agissent sur l'ARNm à différents niveaux, de l'épissage à la traduction. Récemment, un grand nombre d’ARN non-codant des protéines (npcRNAs) ont été identifiés chez les eucaryotes et ont été montré comme interagissant avec une variété de ribonucléoprotéines (RNP) pour contrôler l'expression des gènes au niveau post-transcriptionnel. Nous avons identifié une Nuclear-Speckle RBP (ou NSR) qui interagit avec le npcRNA, ENOD40, un lncARN qui s'accumule au cours de la formation des racines latérales et des nodules chez les légumineuses. Durant cette thèse nous avons analysé le rôle des NSR d’Arabidopsis thaliana ainsi que leur lien avec les npcARN.Deux gènes AtNSRs homologues existent chez Arabidopsis nommés NSRa et NSRb, ces gènes codent des protéines localisées dans des speckles nucléaires avec certaines protéines apparentées à l’épissage. Fait intéressant, les fusions AtNSR-GFP sont relocalisées dans des granules cytoplasmiques dans certaines cellules des racines différenciées ainsi que lors d’une co-expression éctopique de ENOD40. Le gène AtNSRb est régulé par l'auxine alors AtNSRa est constitutif. Les simples mutants Atnsr ne montrent pas de phénotype, mais la croissance des racines des doubles mutants est partiellement insensible à l'auxine, ce qui suggère une fonction redondante de ces protéines dans les racines. La localisation observée pour ces protéines nous a mené à explorer un rôle des NSRs dans l’épissage, nous avons donc analysé le profil d'épissage de 288 gènes en réponse à l'auxine chez Arabidopsis et comparé ces profils entre le WT et les mutants nsra/nsrb. Tout d’abord nous avons remarqué que l’épissage général ne variait pas, en revanche, l’analyse de 288 gènes alternativement épissés montre que le profil d'épissage de 77 gènes semble être modifié durant la réponse à l'auxine et 51 gènes nécessitent les protéines AtNSR pour ce changement. Afin de vérifier l’interaction des NSRs avec les cibles d’AS et avec les npcARN nous avons co-immunoprécipité les NSRs et nous avons identifié au moins 5 cible d’AS et 2 npcARN. L’expression de l’ARN ENOD40 ainsi que du partenaire npcARN module L’AS chez Arabidopsis. Dans un deuxième chapitre, nous avons exploré le rôle des NSRs dans le PTGS déclenché par un transgène contenant un intron ce qui nous a permis de lier l’épissage alternatif et le silencing. Nous proposons donc que les NSRs pourraient lier l’épissage alternatif et l’action des ARN non codants, notamment lors de la croissance de la racine. / In eukaryotes, several RNA binding proteins (RBPs) act on mRNA at various levels from splicing to translation. Recently a large number of non-protein coding RNAs (npcRNAs) have been identified in eukaryotes and shown to integrate into a variety of ribonucleoproteins (RNP) to control posttranscriptional gene expression. Our laboratory has identified a plant Nuclear-Speckle RBP (or NSR) that interacts with an npcRNA, ENOD40 that accumulates during lateral root and nodule formation in legumes. NSR is relocalised into a cytoplasmic RNP in the ENOD40-expressing cells. During this PhD, we have analysed the role of NSRs in Arabidopsis thaliana and its link with npcRNAs. Two AtNSR homologs from Arabidopsis thaliana, named AtNSRa and AtNSRb, code for proteins also localised in nuclear speckles together with certain splicing-related proteins. Interestingly, AtNSR-GFP fusions are relocalised into cytoplasmic granules in certain differentiated root cells and by ectopic expression of the ENOD40 RNA. The AtNSRb gene is regulated by auxin whereas AtNSRa is constitutive. Root growth and lateral root formation of double nsra/nsrb mutants is partially insensitive to auxin. The localisation of these proteins prompted us to explore roles in splicing. No defects in general splicing were observed however analysis of 288 alternatively spliced genes in WT and nsra/nsrb roots in response to auxin revealed 77 changes in splicing profiles in response to auxin from which 51 required AtNSRs. In order to validate the interaction of NSRs with alternatively spliced mRNAs and npcRNAs, we have co-immunoprecipitated NSRs and identified at least 5 interacting alternatively spliced mRNAs and 2 npcRNAs. Expression of the ENOD40 RNA or one interacting ncRNA modulate alternatively splicing in Arabidopsis. In a second chapter, we explored the role of NSRs in the modulation of PTGS triggered by intron-containing transgenes allowing us to link alternatively splicing and silencing. We propose that NSRs may link alternative splicing and the action of non-coding RNA, notably during root growth and development.

Banana bunchy top virus (BBTV) causes one of the most devastating diseases of banana. Transgenic virus resistance is now considered one of the most promising strategies to control BBTV. Pathogen-derived resistance (PDR) strategies have been applied successfully to generate plants that are resistant to numerous different viruses, primarily against those viruses with RNA genomes. BBTV is a circular, single-stranded (css) DNA virus of the family Nanoviridae, which is closely related to the family Geminiviridae. Although there are some successful examples of PDR against geminiviruses, PDR against the nanoviruses has not been reported. Therefore, the aim of this thesis was to investigate the potential of BBTV genes to interfere with virus replication when used as transgenes for engineering banana plants resistance to BBTV. The replication initiation protein (Rep) of nanoviruses is the only viral protein essential for viral replication and represents an ideal target for PDR. Therefore, this thesis focused on the effect of wild-type or mutated Rep genes from BBTV satellite DNAs or the BBTV integral genome on the replication of BBTV in banana embryogenic cell suspensions. A new Rep-encoding satellite DNA, designated BBTV DNA-S4, was isolated from a Vietnamese BBTV isolate and characterised. When the effect of DNA-S4 on the replication of BBTV was examined, it was found that DNA-S4 enhanced the replication of BBTV. When the replicative capabilities of DNA-S4 and the previously characterised Rep-encoding BBTV satellite, DNA-S1, were compared, it was found that the amount of DNA-S4 accumulated to higher levels than DNA-S1. The interaction between BBTV and DNA-S1 was also examined. It was found that over-expression of the Rep encoded by DNA-S1 using ubi1 maize polyubiquitin promoter enhanced replication of BBTV. However, when the Rep-encoded by DNA-S1 was expressed by the native S1 promoter (in plasmid pBT1.1-S1), it suppressed the replication of BBTV. Based on this result, the use of DNA-S1 as a possible transgene to generate PDR against BBTV was investigated. The roles of the Rep-encoding and U5 genes of BBTV DNA-R, and the effects of over-expression of these two genes on BBTV replication were also investigated. Three mutants of BBTV DNA-R were constructed; plasmid pUbi-RepOnly-nos contained the ubi1 promoter driving Rep expression from DNA-R, plasmid pUbi-IntOnly-nos contained the ubi1 promoter driving expression of the DNA-R internal gene product (U5), while plasmid pUbi-R.ORF-nos contained the ubi1 promoter driving the expression of both Rep and the internal U5 gene product. The replication of BBTV was found to be significantly suppressed by pUbi-RepOnly-nos, weakly suppressed by pUbi-IntOnly-nos, but strongly enhanced by pUbi-R.ORF-nos. The effect of mutations in three conserved residues within the BBTV Rep on BBTV replication was also assessed. These mutations were all made in the regions in the ATPase motifs and resulted in changes from hydrophilic to hydrophobic residues (i.e. K187→M, D224→I and N268→L). None of these Rep mutants was able to initiate BBTV replication. However, over-expression of Reps containing the K187→M or N268→L mutations significantly suppressed the replication of BBTV. In summary, the Rep constructs that significantly suppressed replication of DNA-R and -C in banana embryogenic cell suspensions have the potential to confer resistance against BBTV by interfering with virus replication. It may be concluded that BBTV satellite DNAs are not ideal for conferring PDR because they did not suppress BBTV replication consistently. Wild-type Rep transcripts and mutated (i.e. K187→M and N248→L) Rep proteins of BBTV DNA-R, however, when over-expressed by a strong promoter, are all promising candidates for generating BBTV-resistant banana plants.